Membrane fractions of 1,2-sn-diacylglycerol-enriched cells

ABSTRACT

The invention concerns membrane fractions of cells containing a recombinant MGDG synthase and enriched with 1,2-sn-diacylglycerol, their preparation method, their use for screening molecules inducing MGDG synthase activity and a method for screening molecules inducing MGDG synthase activity using said membrane fractions.

The present invention relates to membrane fractions of cells containinga recombinant monogalactosyldiacylglycerol (MGDG) synthase and enrichedin 1,2-sn-diacylglycerol (DAG), to the method of preparing them, totheir use for screening molecules having an effect on MGDG synthaseactivity and to a method of screening molecules having an effect on MGDGsynthase activity, using these membrane fractions.

MGDG is known to be in all plasts analyzed to date: it is the mostabundant lipid of plastid membranes, where it represents more than 50%of glycerolipids. MGDG is vital to plast biogenesis and to cellsurvival, and does not exist in the other membrane systems, inparticular in animal cells (Douce, Sciences, 1974, 183, 852-853); thebiosynthesis thereof is catalyzed in the envelope by a uridine5′-diphosphate galactose (UDP-gal) 1,2-diacylglycerol3-β-D-galactosyltransferase (EC 2.4.1.46) also called MGDG synthase,according to the following reaction:

MGDG synthase is a bifunctional enzyme which binds substrates in anon-ordered manner (Maréchal et al., J. Biol. Chem, 1994, 269,5788-5798), which has oxidation-sensitive cysteines and which isimportant for catalysis (Maréchal et al., J. Biol. Chem., 1995, 270, 11,5714-5722).

MGDG synthase is therefore also an enzyme essential to plast biogenesisand is, consequently, a target of choice for selecting or screeningmolecules with herbicidal potential. In addition, the parasitesresponsible for malaria (4 species of Plasmodium, including P.falciparum), for toxoplasmosis (Toxoplasma gondii) and for scourges ofthe veterinary field, such as coccidiosis (Eimeria) contain degeneratechloroplasts (apicoplasts) which have been demonstrated to be essentialto parasite survival (G. McFadden and David Roos, “Apicomplexan plastidsas drug targets”, 1999, 7, No. 8, 328-333).

The parasites which contain these apicoplasts are called apicomplexanparasites.

A molecule which has an inhibitory action on MGDG synthase activitytherefore has a high herbicide and anti-apicomplexan parasite potentialand can be used advantageously as a novel medicinal product effectiveagainst said apicomplexan parasites or as a herbicide.

The use of an MGDG synthase for selecting or screening productsinhibiting MGDG synthase activity, able to be used as herbicides or asactive principles against apicomplexan parasites has already beenproposed, in particular in patent application FR-A-2 790 915.

According to that application, the selection and/or screening of suchproducts is (are) carried out according to a method comprisingincubating a test substance with an MGDG synthase embedded in biologicalmembranes, and then measuring the specific enzyme activity, after saidincubation.

The biological membranes used in that prior application can inparticular be plastid membranes isolated from plants or else membranefractions of E. coli overexpressing a recombinant MGDG synthase.

Measuring the galactosylation activity carried out by MGDG synthase is,at the current time, very complex and cannot be readily miniaturized, inparticular for the following reasons:

-   -   (1) Addition of two substrates: The two substrates of the enzyme        (DAG and UDP-gal) should be added simultaneously to the        incubation medium. Homogeneity of the system is a problem, in        particular due to the fact that these two substrates have very        different physicochemical properties: one is very hydrophilic        (UDP-gal), the other is very hydrophobic (DAG). The control of        the introduction of these two substrates is therefore difficult        to miniaturize.    -   (2) Addition of a detergent: DAG is so hydrophobic that it is        not water-miscible. In order for the enzyme to have access to        this exogenous substrate, a detergent therefore has to be        introduced into the incubation medium, for example        3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate        (CHAPS), which pulverizes the biological membranes and allows a        rearrangement of all the hydrophobic molecules in the form of        micelles. The micelles are too small to be separated from the        reaction medium by filtration or centrifugation (Stoke's radius        3.8 nm; Maréchal et al, 1994, mentioned above).    -   (3) Phase separation: In order to extract the lipid phase        containing the reaction product (MGDG), a mixture of organic        solvents (chloroform and methanol) is added at the end of the        reaction according to the method described by Bligh et al.        (Can. J. Biochem. Physiol., 1959, 37, 911-917). A biphase forms        and the lipids are recovered in the lower organic phase. The        formation of a biphase and the extraction of an organic phase        are processes which are too sophisticated to be used in a        miniaturized process.

Now, in the context of the search for molecules having an effect on MGDGsynthase activity, it is essential to have a simple, relativelyinexpensive, rapid and miniaturizable method for testing a very largenumber of molecules potentially able to be used as herbicides or asactive principles against apicomplexan parasites.

The inventors have developed the subject of the invention in order toremedy these problems.

Specifically, the inventors have developed new biological membranefractions containing, in the same lipid leaflet, both MGDG synthase andDAG. These membranes can be used in an enzyme method for automated highthroughput screening (HTS) of molecules having an effect on MGDGsynthase activity (inhibitors or activators).

A subject of the present invention is therefore plasma membranefractions from prokaryotic cells or eukaryotic animal cells, consistingof a lipid leaflet containing at least one recombinant MGDG synthase,characterized in that said fractions contain at least 1% by weight ofDAG relative to the total weight of protein, and in that they are in theform of spherical vesicles.

The inventors have in fact demonstrated that the simultaneous presenceof MGDG synthase and DAG in said membrane fractions makes it possible touse these membrane fractions in a method of screening and/or selectingmolecules having an effect on MGDG synthase activity, in which thevolumes of the reaction media can be considerably reduced, thus enablingminiaturization of said method.

By virtue of these membrane fractions, the addition of detergent topromote the control MGDG synthase/DAG mixing is eliminated, and theenzyme reaction takes place directly within the membranes.

According to a preferred embodiment of the invention, the MGDGsynthase/DAG molar ratio is less than 10, and even more particularlyless than 0.12.

Specifically, the enzyme/substrate ratio should respect the conditionsfor measurement making it possible to use the Michaëlis-Mentenenzymological model; in particular, the substrate should not be thelimiting factor of the initial reaction.

The membrane fractions in accordance with the invention are preferablyin the form of spherical vesicles made up of a lipid bilayer.

These membrane vesicles are generally between 0.1 μm and 10 μm in size.

Within these vesicles, the MGDG synthase is, in general, located on theinner face of the lipid bilayer.

These vesicles may be in the form of noninverted, inverted or hybridvesicles.

Disruption of the membranes in fact allows the formation of inverted ornoninverted vesicles; but in both cases, the MGDG synthase is on theface opposite most of the DAG. Fusion between inverted and noninvertedvesicles can generate a new family of vesicles having the MGDG synthaseand the DAG in the same lipid leaflet.

This type of hybrid vesicle can exhibit increased catalytic activity(accessible substrate) and is, consequently, preferred according to theinvention.

A subject of the invention is also a method of preparing membranefractions as described above, characterized in that it consists:

-   -   in a first step, in transforming prokaryotic cells or eukaryotic        animal cells with a construct containing the gene encoding a        plant MGDG synthase,    -   in a second step, culturing said cells in a culture medium which        promotes protein synthesis, so as to induce the synthesis of        MGDG synthase by said cells,    -   in a third step, in incubating the cells cultured in the        preceding step in a reaction medium containing at least one        phospholipase C,    -   and then, in a fourth step, in fractionating the cells thus        enriched in DAG so as to obtain membrane fractions in the form        of spherical vesicles containing at least one recombinant MGDG        synthase and at least 1% by weight of DAG relative to the total        weight of proteins of said membrane fractions.

Among the prokaryotic cells which can be used according to this method,mention may be made of bacteria such as E. coli, which are particularlypreferred according to the invention.

Among the eukaryotic cells which can be used according to this method,mention may be made of cells from yeast, such as Saccharomycescerevisiae, cells from insects, such as drosophila, and also mammaliancells conventionally used to express genes, such as COS cells and CHOcells.

The transformation of the cells in the first step is preferably carriedout on bacterial cells, and more particularly on E. coli cells.

This transformation can be carried out according to the method describedin patent application FR-A-2 790 915, for example by heat shock, with aplasmid pET-Y3a containing the sequence encoding Arabidopsis thalianaMGDG synthase A (Miège et al., Eur. J. Biochem., 1999, 265, 990-1001).

The culture medium used in the second step is a rich culture medium, inorder to promote expression of the MGDG synthase, and is chosen as afunction of the type of cells to be cultured. In the particular case ofbacterial cells, and in particular of E. coli, Luriat Broth (LB) mediumcan be used.

The use of PLC during the third step of the method of preparation inaccordance with the invention is essential to the enrichment of theplasma cell membranes in DAG.

Specifically, these cell membranes are rich in phospholipids,particularly in phosphatidylethanolamine (80% of phospholipids). Thesephospholipids can be hydrolyzed by PLC, which is not specific for thepolar head.

PLC catalyzes the conversion of phosphatidylethanolamine to DAGaccording to the following reaction:

PLC therefore makes it possible to enrich the cell membranes in DAG byhydrolysis of its phospholipids.

The nature of the PLC used according to the invention is not critical,on condition that it is active on the phospholipids of the membranesintended to be enriched in DAG.

According to a particularly preferred embodiment of the invention, aBacillus cereus phospholipase C is used.

The PLC is preferably used at a concentration between 1 U and 20 U perml of reaction medium, and even more preferentially at a concentrationbetween 5 and 12 U/ml.

Of course, the reaction medium used in the third step is generally abuffer medium the pH of which should be compatible with the correctfunctioning of the PLC. This pH is generally between 6 and 8.

In the fourth step, the membranes are fractionated so as to generatemembrane fractions which close up spontaneously in the form of membranevesicles. The fractionation of the membranes can be carried out bymechanical shock, for example using a French press, by thermal shock(freezing/thawing), or by osmotic, electric or else physical shock, suchas by sonication.

The membrane fractions thus obtained can then optionally be purified,for example on a cushion of Percoll.

When the preparation of the membrane fractions in accordance with theinvention is finished, they can optionally be frozen before being usedfor selecting or screening molecules having an effect on MGDG synthaseactivity.

A subject of the invention is therefore also the use of the membranefractions as described above, for selecting and/or screening moleculeshaving an effect on MGDG synthase activity.

In particular, a subject of the invention is the use of the membranefractions as described above, for selecting or screening moleculesinhibiting MGDG synthase activity, able to be used as active principlesagainst parasites or as herbicides.

A subject of the invention is also a method of selecting and/orscreening molecules having an effect on MGDG synthase activity,characterized in that it comprises:

-   -   a step comprising incubation of the test substance(s) with a        sufficient amount of membrane fractions as defined above and of        radiolabeled UDP-galactose in an aqueous phase having a pH of        between 4 and 11,    -   a step comprising washing of the membrane fractions,    -   a step comprising separation of the membrane fractions,    -   then a step comprising determination of the MGDG synthase        activity.

According to this method, the enzyme reaction takes place directlywithin the membrane fractions, the size of which is compatible withseparation from the reaction medium by centrifugation or by filtration.Using this means, it is no longer necessary to use organic solvent toextract the reaction products, the MGDG in fact being trapped in themembrane fractions on which a simple measurement of the radioactivity ofthe radiolabeled galactose incorporated can be carried out.

According to a preferred embodiment of the invention, the aqueousincubation phase contains a buffer and has a pH of between 6 and 8.

By way of example, the buffer may in particular be3-(N-morpholino)propanesulfonic acid or a KCl/K₂HPO₄ mixture.

The amount of UDP-galactose to be used in the incubation medium should,of course, be sufficient so as not to constitute a limiting factor ofthe reaction. This amount is preferably between 0.1 and 10 nmol per μlof reaction medium.

The incubation step is preferably carried out at ambient temperature,for a period of at least 10 seconds, and even more particularly for aperiod of between 1 and 45 minutes.

At the end of the incubation step, the reaction is preferably stopped bycooling the incubation medium (in general to a temperature ofapproximately 4° C.) or by centrifugation.

When the reaction is finished, the step comprising washing of themembrane fractions is carried out in order to eliminate the excessradiolabeled UDP-galactose which has not been incorporated by themembrane fractions. One or more successive washes may be carried out,generally with water.

The membrane fractions are then separated by centrifugation or byfiltration, the latter technique being particularly suitable forminiaturization of the method on microplates.

The determination of the MGDG synthase activity is carried out bymeasuring the amount of radiolabeled galactose incorporated into themembrane fractions. This measurement is conventionally carried out usinga radioactivity counter.

The method of selecting and/or screening molecules having an effect onMGDG synthase activity in accordance with the invention can be readilyminiaturized since it uses small reaction volumes and does not usedetergents or organic solvent, as is the case in the methods previouslydescribed in the prior art.

A subject of the invention is also therefore microtitration platescomprising a multitude of wells, characterized in that the bottom of thewells consists of a filter and in that said wells contain membranefractions as described above.

These microplates can be stored in a freezer before use. They canoptionally be equipped with a detachable bottom.

The microplates in accordance with the invention make it possible todetermine the MGDG synthase activity by direct measurement of theradioactivity of the radiolabeled galactose incorporated into themembrane fractions retained by the filter at the bottom of the wells.

They may therefore be used to test large “chimiotheques” [chemicallibraries] of molecules for their activity of an effect on MGDG synthaseactivity.

Finally, a subject of the invention is the use of at least one moleculeinhibiting MGDG synthase activity as selected in accordance with themethod of screening and selecting in accordance with the invention, forpreparing an antiparasitic medicinal product or a herbicide.

Besides the arrangements above, the invention also comprises otherarrangements which will emerge from the following description, whichrefers to an example of preparation of membrane fractions in accordancewith the invention, to a comparative example of determination of MGDGsynthesis activity, to an example of demonstration of the presence ofMGDG in an apicomplexan parasite, and to the attached figures, in which:

FIG. 1 represents the number of nmole of galactose incorporated per hourinto membrane fractions enriched in DAG as a function of the number ofμg of proteins,

FIG. 2A represents the number of nmole of galactose incorporated perhour and per mg of proteins (membrane fractions enriched in DAG) as afunction of the number of μmole of DAG,

FIG. 2B represents an inverse coordinate plot of the Line Weaver andBurk type on which the inverse of the number of μmoles of galactoseincorporated per hour and per mg of proteins is expressed as a functionof the inverse of the number of μmoles of DAG;

FIG. 3 represents the amount of labeled galactose incorporated by themembrane lipids of Toxoplasma gondii.

It should be clearly understood, however, that these examples are givenonly by way of illustration of the subject of the invention, of whichthey in no way constitute a limitation.

EXAMPLE 1 Preparation of Membrane Fractions Containing an MGDG Synthaseand DAG

1) Production of Recombinant MGDG Synthase in E. coli

A—Transformation of the Bacteria

The bacteria are transformed according to the method described in patentapplication FR-A-2 790 915.

All the cultures are prepared under sterile conditions. Competentbacteria (bacterial strain BL21 or BLR of Escherichia coli) aretransformed by heat shock with a plasmid pET-Y3a which makes it possibleto overcome the problem due to the fact that the deduced sequence of theMGDG synthase contains 22 arginine residues, among which 17 are encodedby AGG or AGA, codons which are in fact used very little in E. coli.

The plasmid pET-Y3a has been described in patent application FR-A-2 790915 and is constructed by inserting the arg U gene (or DNA Y) encodingthe arginine transfer RNA associated with the rare codons AGA/AGG, intothe plasmid pET-3a (Novagen). The plasmid pET-Y3a contains the sequenceencoding Arabidopsis thaliana MGDG synthase A (under the control of apromoter inducible with isopropyl-β-D-thiogalactopyranoside: IPTG), acarbenecillin resistance gene and a sequence encoding the argininetransfer RNA. This ARG4 tRNA allows the synthesis of proteins such asMGDG synthase, the sequence of which contains many Arg codons, which arerare in bacteria.

B—Production of Recombinant MGDG Synthase A in E. coli

A colony isolated from recombinant bacteria is transferred into 8 ml ofLuria Broth (LB) medium in the presence of antibiotic (20 μg/ml finalconcentration of carbenecillin). The preculture is incubated at 37° C.,with regular shaking, and the evolution of bacterial growth is followedby measuring the optical density (OD) at 600 nm, until a value of 0.5 isobtained.

The preculture is transferred into 500 ml of LB medium (20 μg/ml finalconcentration of carbenecillin) and incubated at 37° C. with regularshaking. At an OD measured at 600 nm of 0.5, the bacterial population isthen in its exponential growth phase, which is a time of intense proteinsynthesis.

The addition of IPTG (0.4 mM final concentration) makes it possible toinduce synthesis of the recombinant MGDG synthase.

The culture is then incubated for 3 hours with shaking at 28° C. inorder to promote production of the protein in its active form.

The suspension of induced bacteria is divided up into two fractions of250 ml and subjected to centrifugation for 15 minutes at 5 000 rpm(Sorvall® RCSC centrifuge, GS-3 rotor).

The recovered pellets are resuspended in 10 ml of culture medium (LB,carbenecillin at 20 μg/ml final concentration) and centrifuged for 15minutes at 5 000 rpm (Sorvall® RCSC, SLA 600 TC rotor).

The supernatant is removed and the bacterial pellet is stored at −80° C.

2) Enrichment of the Bacterial Membranes in DAG by Treatment withPhospholipase C

5 ml of pellet of the bacteria induced for MGDG synthase in the previousstep are resuspended in 5 ml of 10 mM 3-(N-morpholino)propanesulfonicacid (MOPS), pH 7.8, 1 mM dithiothreitol (DTT) and 10% (w/v)glycerol.

20 μl of Bacillus cereus phospholipase C (PLC) (phosphatidyl-cholinecholinephosphohydrolase, EC 3.1.4.3) are added in order to obtain afinal concentration of 8 U/ml. The reaction is carried out at ambienttemperature for 3 hours with stirring and is stopped by adding EDTA (0.4M final concentration).

3) Purification of Bacterial Membranes

A solution of the induced bacteria, the membranes of which are enrichedin DAG by treatment with PLC, as obtained above in the previous step, istaken up in 50 ml of 5 mM MOPS, pH 7.8, 0.5 mM DTT, 5% (w/v)glycerol.

The bacteria are ruptured at high pressure (800 psi) using a Frenchpress: the membranes in aqueous solution then organize themselvesspontaneously into vesicles and into inverted vesicles.

6 ml of the lysate obtained are deposited on a cushion of 35% Percoll(in 10 mM MOPS, pH 7.8, 1 mM DTT, 10% glycerol) and then centrifuged for10 minutes at 5 000 rpm, at a temperature of 4° C. (Sorvall® RC SC, HB-6rotor).

Vesicles consisting of a lipid bilayer containing MGDG synthase and atleast 1% by weight of DAG relative to the total weight of protein areobtained.

Four fractions are then formed: a supernatant (in the upper part of thetube), the supernatant/Percoll interface, the cushion of 35% Percoll anda pellet.

All the membrane fractions are collected and washed by dilution with 5volumes of 10 mM MOPS, pH 7.8, 1 mM DTT, 10% (w/v) glycerol, andcentrifuged for 15 minutes at 5 000 rpm at a temperature of 4° C.(Sorvall® RCSC, HB-6 rotor).

The supernatants are removed and the pellets, which are fragile, arecarefully taken up in 1 ml of washing medium so as to be centrifugedagain for 10 minutes at 13 000 rpm at a temperature of 4° C. (Eppendorffcentrifuge 5804).

The pellets are resuspended in 500 μl of 10 mM MOPS, pH 7.8, 1 mM DTT,10% (w/v)glycerol and are stored at −20° C.

EXAMPLE 2 Comparative Determination of MGDG Synthesis Activity Accordingto the Prior Art and According to the Invention

I—Conventional Measurement of the MGDG Synthesis Activity According tothe Method of Bligh and Dyer (1959)

This method was described in the article by Bligh and Dyer, “A RapidMethod Of Total Lipid Extraction and Purification”, Can. J. Biochem.Physiol., 1959, 37, 911-917.

The measurement of the galactosylation activity is based onincorporation of the galactose originating from the radioactive (¹⁴C)UDP-gal into the lipid fraction of the reaction medium.

The reaction is carried out at ambient temperature. 100 μg of DAGhydrophobic substrate (1 mg/ml) and 200 μg of phosphatidylglycerol (PG)in solution in chloroform (10 mg/ml) are introduced into a tube, driedunder argon and then resuspended with 11 μl of a detersive medium (85 mMCHAPS, 0.7 M MOPS, 14 mM DTT), 75 μl of KCl (1 M) and 75 μl of KH₂PO₄ (1M).

The presence of the detergent, in this case CHAPS, makes it possible tocreate mixed micelles containing the detergent, the DAG, the PG and theMGDG synthase originating from the sample.

After addition of 150 μl of sample, the final composition of thereaction medium (50 mM MOPS, 1 mM DTT, 250 mM KCl, 250 mM KH₂PO₄, 6 mMCHAPS in 300 μl of final volume) satisfies the criteria of the surfacedilution model (Maréchal et al, 1995 mentioned above).

This example uses CHAPS as detergent, but it is also possible to useother detergents such as, for example, cholate, deoxycholate,Triton-X100®, NONIDET®, octylglucoside or lauryldimethylamine oxide(LDAO).

The reaction is started by introducing 10 μl of UDP-[¹⁴C]-galactose (NewEngland Nuclear 25 Bq/μmol, 10 mM). The reaction is stopped by adding1.5 ml of a ½ (v/v) chloroform/methanol mixture.

The addition of 0.5 ml of chloroform and of 0.6 ml of water makes itpossible, after centrifugation for 10 minutes at 1 000 rpm (EppendorffA-4-44 centrifuge, 5804 rotor), to obtain a distinct biphase (a highlyradioactive aqueous phase in the upper part of the tube, and an organicphase in the lower part).

The aqueous phase contains the radioactivity of the residual UDP-gal notused by the enzyme, while the organic phase contains the radioactivityof the hydrophobic product of the reaction: the MGDG.

After two washes of the aqueous phase with an identical volume ofaqueous phase without UDP-[¹⁴C]-gal, the organic phase containing theMGDG produced is transferred into a counting vial.

The fraction recovered is dried under argon and taken up in 10 ml ofscintillation fluid, and the radioactivity of the sample is estimated.

The galactosylation activity is defined by the number of μmol ofgalactose incorporated into the lipid fraction per mg of protein and perhour.

II—Measurement by Purification of the Membranes after Centrifugation inAccordance with the Invention

This measurement can be carried out only for a sample consisting ofvesicles of membranes sequestering both the enzyme (MGDG synthase) andits hydrophobic substrate (DAG). Then only in this case, it is no longernecessary to add a detergent so that enzyme and substrate come intocontact.

The reaction medium used is different from that given in theconventional method after extraction of the lipids with organicsolvents.

The membrane fraction sample containing the MGDG synthase and the DAG,as obtained above in example 1, step 3, is suspended in a final volumeof 300 μl containing 250 mM KCl and 250 mM KH₂PO₄.

The reaction is started by adding UDP-radiolabeled [¹⁴C]gal.

The reaction is stopped by transferring to ice for 10 minutes.

Washing of the excess UDP-[¹⁴C]gal (not incorporated into the membranes)is carried out by centrifugation of the sample for 10 minutes at 13 000rpm at a temperature of 4° C., and taking up the pellet in 500 μl ofsterile water.

This washing step is repeated three times. The pellet obtained is driedby centrifugation under vacuum for 1 hour (Eppendorff Concentrator 5301Speed-vac). The dry sample is then taken up in a ½ (v/v)chloroform/methanol mixture, in order to transfer it into a countingvial, dried under argon, and solubilized with 10 ml of scintillationfluid.

The radioactivity of the sample is estimated using a Kontron®(Betamatic) counter.

The galactosylation activity is defined by the number of μmole ofgalactose incorporated per mg of protein and per hour.

III—Polyacrylamide Gel Electrophoresis Under Denaturing Conditions

The samples to be analyzed are suspended in the loading medium (0.15 MTris HCl, pH 6.8, 10% glycerol, 0.02% SDS, 0.01% bromophenol blue,0.025% DTT), and are then boiled for 4 minutes.

The acrylamide solutions are prepared in a 25 mM Tris buffer (pH 8.3 inthe separating gel and pH 6.5 in the stacking gel) containing 0.192 Mglycine and 0.1% sodium dodecyl sulfate (SDS).

The electrophoresis on a stacking gel (5% acrylamide) and then on aseparating gel (12% acrylamide) is carried out at ambient temperature ina 25 mM Tris buffer containing 0.192 M glycine (pH 8.3) and 0.1% (w/v)SDS (U.K. Laemmli, Nature, 1970, 227, 680-683), under a constant voltageof 100 V.

The migration is stopped when the bromophenol blue leaves the gel.

The proteins are then stained with Coomassie blue (0.5% (w/v) Coomassiebrilliant blue 8250, 25% methanol, 10% (v/v) acetic acid).

The gel is destained with successive baths of 25% isopropanol, 10% (v/v)acetic acid.

IV—Assaying of Proteins

Principle: the proteins are assayed by the Lowry method (Lowry et al.,J. Biol. Chem., 1951, 193, 265-275), by measuring two simultaneouscolored reactions.

A first reaction similar to the “biuret” reaction leads to the formationof a complex between the peptide bonds of the proteins (—CO—NH—) and theCu²⁺ ions in alkaline medium, and a second reaction leads to reductionof the Folin-ciocalteu reagent by the phenols of the tyrosines. Thismethod makes it possible to assay solutions having a concentrationranging from 2 to 200 mg/ml.

Experimental approach: the volume of the sample to be assayed isadjusted to 200 μl with sterile water then 1 ml of assay reagentprepared extemporaneously (50 volumes of 2% CO₃Na₂, 0.1 N NaOH+1 volumeof CuSO₄, 0.5% SH₂O, 1% sodium tartrate).

After reaction for 10 minutes at 20° C., 100 μl of Folin-ciocalteureagent are added and the reaction is incubated at 20° C. for 30minutes.

The amount of proteins in the sample to be assayed is determined bycomparison of absorbence at 750 nm with a standard range establishedwith bovine serum albumin (BSA).

V—Results

A—The Recombinant Bacteria must be Artificially Enriched in DAG in Orderto Allow Measurement of the Galactosylation Activity in the Presence ofUDP-Gal

Analysis of the total proteins of bacterial samples taken during theinduction step (example 1, step 1, FIG. 1) shows that induction withIPTG leads to an accumulation of MGDG synthase A representingapproximately 30% of the proteins.

A comparison of the galactosylation activity measured in a crude extractof induced bacteria expressing the MGDG synthase A in the presence andin the absence of DAG was carried out.

The galactosylation activity was measured by extraction of the lipidsaccording to the method of Bligh and Dyer (see above) on 20 μl of abacterial culture induced at 28° C., in the absence of PG and possiblyplaced in the presence of 50 μg of DAG.

The results obtained appear in table I below:

TABLE I Incubation of bacteria in 270 μM MGDG synthase activity (innmole of of DAG galactose incorporated per hour) yes 576 no 5

These results show that a considerable galactosylation activity isobserved in this extract incubated with 270 μM of DAG, whereas there isvirtually no incorporation of galactose into the bacterial membranesincubated without the exogenous introduction of DAG.

Consequently, the bacterial membranes do not have a sufficient amount ofendogenous DAG to carry out an isolated measurement of the activity ofthe recombinant enzyme.

B—Treatment of the Bacterial Membranes with PLC Generates DAG Availablefor Measuring Galactosylation Activity

The galactosylation activity of the MGDG synthase was measured aftersynthesis of endogenous DAG by PLC or after addition of exogenous DAG.This activity was measured on 20 μl of the same bacterial cultureinduced at 28° C., in the presence or absence of 3 μl of PLC (2 000U/ml), of 50 μg of DAG (at 1 mg/ml and optionally in the presence of 1or 10 μl of CaCl₂.

The results are given in table II below:

TABLE II DAG in μM PLC CaCl₂ in μM Galactose incorporated (nmol/hour) —— — 7 270 — — 30 — + — 91 270 + — 99 270 + 33 118 270 + 330 109 — + 3394 — + 330 97

These results show that treating the bacterial membranes with PLC makesit possible to measure a galactosylation activity greater than thatobtained after adding 270 μM of exogenous DAG.

The PLC therefore makes it possible to load the bacterial membranes withDAG by hydrolysis of its phospholipids. The poor incorporation ofgalactose obtained when adding DAG is explained by the fact that a verysmall proportion of added DAG was able to penetrate into the bacterialmembranes in the absence of PLC, in order to act as substrate for theMGDG synthase.

It is also important to note that the calcium, which is an activator ofPLCs, has no effect on the measured galactose incorporation, suggestingthat the concentration of Ca²⁺ of the bacterial suspension is sufficientto measure optimal activity of the PLC.

C—Incorporation of Radioactive Galactose into Membranes Enriched in DAG

The incorporation of radioactive galactose into the lysed membranevesicles as described above in example 1, step B-3) was measured andcompared to the incorporation of radioactive galactose by bacteriainduced to express MGDG synthase A, but not lysed. The galactosylationactivity is measured on 150 μl of sample (as described previously) inthe presence or in the absence of 150 μg of PG (at 10 mg/ml) and of 100μg of exogenous DAG (at 1 mg/ml).

The results obtained are given in table III below:

TABLE III Galactose incorporated (nmol/hour) Induced bacteria, Withoutexogenous +550 μM of exogenous treated with PLC introduction of DAG DAGand 667 μM of PG Not lysed 2 426 6 622 Lysed 3 172 8 663

These results show that the galactosylation activity measured on lysedbacteria without the addition of DAG or PG indicates that vesiclescontaining both the MGDG synthase and the DAG have been formed, and thatthe enzyme has conserved its catalytic capacity.

They also show that the activity of the lysed fractions is greater thanthat of the non lysed fractions. This observation is coherent with theMGDG synthase A being located on the inner face of the lipid bilayer ofthe bacterial membranes. Specifically, since the PLC only has access tothe outer lipid bilayer, the fraction of MGDG synthase located on theinner face can capture only a portion of the DAGs formed which haveundergone transverse displacement from the outer leaflet to the innerleaflet of the membrane. The disruption of the membranes allows theformation of inverted and noninverted vesicles, but, in both cases, theMGDG synthase is on the face opposite most of the DAG. Fusion betweeninverted and noninverted vesicles may generate a new family of vesicleshaving, in the same membrane leaflet, the enzyme and the DAG. This typeof vesicle may exhibit increased catalytic activity (accessiblesubstrate). If it is supposed that the generation of these threepopulations of vesicles, inverted, noninverted and hybrid, is equallyprobable, then the activity of the lysed samples is increased by afactor of 1.33 compared to the non lysed samples, which is observed inthe present case.

D—Development of an Assay which can be Miniaturized

1) Demonstration of the Role of PLC

A suspension of lysed bacteria containing membrane vesicles enriched inMGDG synthase A and optionally in DAG by treatment with PLC isfractionated on a gradient of 35% Percoll (9 ml) as previouslydescribed. The fractions corresponding to the supernatant and to theinterface (S+SL: 6 ml) and also those corresponding to the pellet (P: 1ml) are collected. The galactosylation activity and the amounts ofproteins are measured on the various fractions sampled (S+SL=6 ml, P=1ml) and also of the fraction deposited (D of 6 ml).

The results obtained are given in table IV below:

TABLE IV Total activity Specific (in nmol activity (nmol UDP-gal UDP-galProteins in incorporated/ incorporated/ Enrich- Fractions μg/ml hour)hour/mg) ment No D 400 103  43 1 treatment S + SL 136 363 445 10.3 withPLC P 39  3  83 1.9 Treatment D 500 618 206 1 with PLC S + SL 880 11 4622 169 10.5 P 70  67 955 4.6

These results show that the membranes which have undergone enrichment inDAG by treatment with PLC are capable of converting much more UDP-galthan the membranes not enriched in DAG, since they have not undergoneany treatment with PLC.

Analysis of the fractions by acrylamide gel electrophoresis underdenaturing conditions shows enrichment for a polypeptide correspondingto MGDG synthase, from the supernatant to the pellet. The lowgalactosylation activity in the pellet (table IV) suggests an enrichmentof this fraction mainly in inclusion bodies (inactive enzyme). The S+SLfractions containing the MGDG synthase A associated with the membranesenriched in DAG were therefore selected in order to analyze the enzymeactivity of the MGDG synthase as a function of the criteria for validityof Michaelis-Menton enzymology.

2) Analysis of the Enzyme Activity of the MGDG Synthase

The results are given in FIG. 1, which represents the initial rate (innmol of galactose incorporated per hour) as a function of the number ofμg of proteins/tube. The initial rates were determined by measuring theincorporation of galactose after 15 and 30 minutes incubation of variousamounts of the membranes treated with PLC and present in the treatedS+SL fractions (25, 50, 75 μg of proteins).

This FIG. 1 shows that the incorporation of galactose is a linearfunction of the amount of sample incubated in the presence of UDP-gal.The slope of the curve makes it possible to deduce an activity of 340nmol of galactose incorporated per hour and per mg of proteins.

The amount of DAG obtained after treatment with PLC is undetermined butthe concentration of this substrate in the membrane vesicles remainsconstant whatever the amount of proteins of the sample. In this respect,the surface concentration of DAG (in molar fraction or in mol of DAG perunit of membrane surface) and the molar concentration of UDP-gal areconstant. As a result, the initial reaction rate is directlyproportional to the amount of proteins, showing the limiting nature ofthe amount of enzyme. The relationship of linearity is a condition ofvalidation of conditions for Michaelis-Menton enzymological analysis.

3) Evaluation of the Amount of DAG Generated in the Bacterial Membranesby Treatment with PLC

The activity (in number of nmol of galactose incorporated/hour/mg) of 5μg of membranes not treated with PLC and purified on a Percoll gradientwas measured for varying amounts of DAG introduced (FIG. 2A). Theinitial rates were determined by measuring the incorporation ofgalactose after 15 and 30 minutes of incubation. The measurements weremade for varying amounts of DAG introduced (12.5 μg; 25 μg; 37.5 μg; per300 μl of reaction medium).

The measurements make it possible to establish an inverted coordinateplot of the Lineweaver and Burk type (FIG. 2B).

If it is considered that the measurements made on membranes not treatedwith PLC constitute standard curves to estimate a DAG content, a sampleof 5 μg, the intrinsic activity of which is 340 nmol/h/mg, is equivalentto the same untreated sample to which 0.5 μg of DAG has been added.

It is therefore possible to deduce therefrom that the treatment with PLCmakes it possible to obtain about 0.1 μg of DAG per μg of proteins.

4) Measurement of the Galactosylation Activity of Membranes Enriched inMGDG Synthase and in DAG before and after Thawing

The ability of membranes having undergone freezing for one day at −20°C. to incorporate galactose was measured and compared to that ofmembranes which had not undergone any freezing step.

No significant difference was observed between the two samples.

Consequently, this property makes it possible to prepare the sample ofmembranes and to manipulate it reproducibly in an automated device forscreening molecules having an effect on MGDG synthase activity, whichmay comprise steps consisting of storing the sample under coldconditions, without loss of activity.

5) Simplified Measurement of the Galactosylation Activity Contained inthe Sample of Membranes Enriched in MGDG Synthase and in DAG

Membrane vesicles containing MGDG synthase enriched in DAG by treatmentwith phospholipase C, and optionally purified, can therefore be used tomeasure the production of MGDG in these same vesicles.

50 μg of membrane vesicles (equivalent to 50 μg of MGDG synthase and 50μg of DAG) are suspended in an aqueous phase (250 mM KCl, 250 mM K₂HPO₄,300 μl final volume, pH 7.8) contained in a tube (tube No. 1).

By way of comparison, a control tube (tube No. 2) was prepared: 50 μg ofMGDG synthase and 50 μg of DAG were suspended in the same aqueous phaseas that used in tube No. 1.

The reaction is initiated in each tube by adding 10 μl of UDP-galradiolabeled with ¹⁴C on the galactose, and then stopped after 30minutes by cooling to 4° C.

The reaction medium of tube No. 1 is then centrifuged at 13 000 rpm for10 minutes, rinsed several times with 500 μl of water, then centrifugedagain at 13 000 rpm for 10 minutes.

Tube No. 2 was treated using the conventional method of lipid extractionwith organic solvents, as described previously.

The radioactivity recovered in the centrifugation pellet (tube No. 1)was measured using a radioactive counter and was 4 026 dpm.

The radioactivity measured after extraction of the lipids in tube No. 2was measured in the same way and was 4 136 dpm.

Consequently, the radioactivity measured for each of the tubes is notsignificantly different and makes it possible to validate the method ofmeasuring the MGDG synthase activity in accordance with the invention.

The sample of membrane vesicles prepared in accordance with theinvention contains the enzyme and its hydrophobic cosubstrate underconditions which correspond to measurement of the enzymological activityby the Michaelis method. In addition, this sample allows simple andminiaturizable measurement of the galactosylation activity bycentrifugation and, by extension, by filtration.

VI—Conclusion

This example demonstrates that it is possible to use membrane fractionsderived from a culture of cells expressing a recombinant MGDG synthaseto measure a galactosylation activity respecting Michaelis-Menton laws,and the procedure for the measurement of which can be miniaturized onmicroplates.

It has also been demonstrated that the incorporated radioactivity can besimply recovered in the aggregates of the reaction medium, collected bycentrifugation. Thus, the MGDG generated by this system accumulates inthe membranes that a system of measurement by microfiltration issufficient to measure, making it possible to use this enzyme assay forhigh throughput screenings (HTS) of active molecules. In particular, itis now possible to screen a “chimiothèque” [chemical library] by thismethod in order to select novel molecules with herbicidal potential. Theinhibitors specific for MGDG synthase A thus produced will, moreover, bepowerful pharmacological tools for allowing physiological studies ofgalactolipid synthesis.

EXAMPLE 3 Demonstration of MGDG Synthesis in an Apicomplexan Parasite

The aim of this example is to demonstrate the presence of MGDG in anapicomplexan parasite, Toxoplasma gondii and that, consequently, theMGDG synthase which serves as a target to search for a molecule withantiparasitic properties clearly exists in apicomplexans.

2×10⁸ cells of Toxoplasma gondii, in the form of tachyzoites, aresuspended in 0.1 ml of a 1:10 mixture of 10 mM3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.8 and 1 mMdithiothreitol (DTT), containing 2% (w/v) glycerol and 50 mM of KCl, andthen incubated for 30 min in the presence of 4 μCi of UDP-[³H]-galactose(7.5 nmol).

The glycolipids are extracted according to the method of Bligh and Dyer,1959 (mentioned above), then analyzed by thin layer chromatography (TLC)on 60μ silica gel plates resolved with a 65/25/4 (v/v)chloroform/methanol/water mixture, in the presence of control lipids(MGDG; bovine brain monogalactosyl cerebroside (MGCB);digalactosyldiacylglycerol (DGDG); trigalactosyldiacylglycerol (triGDG)and tetragalactosyldiacylglycerol (tetraGDG)).

The radioactivity of the labeled lipid is then detected using aTLC-analyzer device (LB2842 automatic TLC scanner).

The results obtained are given in FIG. 3, which illustrates the amountof labeled galactose (cpm) incorporated by the Toxoplasma gondii cellsas a function of migration in centimeters.

On this figure, it is possible to see 3 first peaks which migrate to thesame degree as the MGCB, whereas the last peak migrates to the samedegree as the MGDG.

After migration, the lipids are visualized by spraying, onto the silicagel plates, a solution comprising 0.2% of orcinol and 75% of sulfuricacid, and then heating at a temperature of 100° C. for 15 minutes(results not given).

The peak corresponding to the MGDG disappears after alkali hydrolysisfor 3 hours with 0.1 N potassium hydroxide in a water/methanol mixture.

Complete identification of the lipids is carried out after hydrolysis ofthe polar head with α-galactosidase from green coffee beans andβ-galactosidase from bovine testes, and deacylation by alkali hydrolysisunder gentle conditions (0.1 N KOH in a water/ethanol mixture for 3hours).

Peak 4 is sensitive to hydrolysis with β-galactosidase, which shows thatthe galactose is clearly linked in the beta position, as for MGDG.

Moreover, after alkali hydrolysis, peak 4 disappears, which demonstratesthat the lipid present in the Toxoplasma gondii membrane clearlycontains half diacylglycerol.

This experiment demonstrates the existence of MGDG in the membrane ofToxoplasma gondii tachyzoites and confirms that an application of thesearch for inhibitors of plant MGDG synthase is the identification ofanti-apicomplexan parasite agents.

1. A bacterial plasma membrane fraction comprising a lipid leafletcontaining a recombinant plant monogalactosyldiacylglycerol (MGDG)synthase from Arabidopsis thaliana, wherein said fraction contains atleast 1% by weight of diacylglycerol (DAG) relative to the total weightof protein, and further wherein said fraction comprises sphericalvesicles, the membrane of said spherical vesicles comprising a lipidbilayer.
 2. The fraction as claimed in claim 1, wherein the MGDGsynthase/DAG molar ratio is less than
 10. 3. The fraction as claimed inclaim 1, wherein the MGDG synthase/DAG molar ratio is less than 0.12. 4.The fraction as claimed in claim 1, wherein the membrane vesicles arebetween 0.1 μm and 10 μm in size.
 5. The fraction as claimed in claim 1,wherein the MGDG synthase is located on the inner face of the lipidbilayer.
 6. The fraction as claimed in claim 1, wherein the vesicles arein the form of noninverted, inverted or hybrid vesicles.
 7. The fractionas claimed in claim 6, wherein the vesicles are in the form of hybridvesicles comprising the MGDG synthase and the DAG in the same lipidleaflet.
 8. A method of preparing the bacterial plasma membrane fractionas defined in claim 1, comprising: in a first step, transformingbacteria with a construct containing the gene encoding the recombinantplant MGDG synthase, wherein said plant is Arabidopsis thaliana in asecond step, culturing said bacteria in a culture medium which promotesprotein synthesis, so as to induce the synthesis of the recombinantplant MGDG synthase by said bacteria, in a third step, incubating thebacteria cultured in the preceding step in a reaction medium containingphospholipase C, and in a fourth step, fractionating the bacteria so asto obtain the plasma membrane fraction comprising spherical vesiclescontaining said recombinant plant MGDG synthase and at least 1% byweight of DAG relative to the total weight of protein of said membranefraction.
 9. The method as claimed in claim 8, wherein a Bacillus cereusphospholipase C is used.
 10. The method as claimed in claim 8, whereinthe phospholipase C is used at a concentration between 1 U and 20 U perml of reaction medium.
 11. The method as claimed in claim 8, wherein thefractionation of the membranes is carried out by mechanical shock,thermal shock, osmotic shock, electric shock or by physical shock. 12.Microtitration plates comprising a multitude of wells, wherein thebottom of the wells consists of a filter and further wherein said wellscontain a membrane fraction as defined in claim 1.